Structure, Function and Regulation of Renal K Channels

我们的实验室专注于肾钾通道的研究，这是一组多样的膜蛋白，有助于K在细胞膜上的被动（由K的电化学梯度驱动）运动。这项工作导致了两个新的K通道基因的发现，KCNA10（振荡器相关的，电压门控K通道），KCNK1（一个双孔通道）和获得的功能突变的电压门控振荡器K通道，KCNA3。KCNA10是本发明的主题，其编码在肾脏、心脏和血管中表达的电压门控、环核苷酸调节的K选择性通道。我们的主要目标是确定其分子结构并确定其在肾脏中的作用。初步研究表明，KCNA10是一种异源多聚体复合物，这一点可以通过鉴定与KCNA10相互作用并改变其动力学特性和表达水平的三种蛋白质来证明。我们建议调查这些相互作用的分子机制。此外，我们计划确定我们最近发现的KCNA10剪接异构体的动力学特性和肾脏表达。用免疫细胞化学方法检测到KCNA10蛋白在大鼠近曲小管腔膜上的表达。Western blotting也证实了KCNA10蛋白在大鼠皮质膜中的表达。基于上述，我们假设KCNA10是一个异源多聚体，电压门控，顶端钾通道，调节近端肾小管膜电位。因此，我们拟采用膜片钳技术证实KCNA10在近端小管顶膜的表达，建立KCNA10基因敲除小鼠模型，并对其进行全面的研究，特别关注其对近端小管功能的作用。这些研究应该提供重要的信息，关于KCNA10的分子结构及其在肾脏溶质稳态中的作用。

Our laboratory is focused on the study of renal potassium channels, a diverse group of integral membrane proteins that facilitate the passive (driven by the electrochemical gradient for K) movement of K across the cell membranes. This work has led to the discovery of two novel K channel genes, KCNA10 (Shaker-related, voltage-gated K channel), KCNK1 (a two-pore channel) and to a gain-of-function mutation in the voltage-gated Shaker K channel, KCNA3. KCNA10, the subject of the present proposal, encodes a voltage-gated, cyclic nucleotide regulated, K-selective channel expressed in kidney, heart and blood vessels. Our main goals are to determine its molecular structure and establish its role in kidney. Preliminary studies suggest that KCNA10 is a heteromultimeric complex as evidenced by the identification of three proteins that interact with KCNA10 and modify its kinetic properties and expression level. We propose to investigate the molecular mechanisms underlying these interactions. In addition, we plan to determine the kinetic properties and renal expression of a KCNA10 splice isoform we recently identified. KCNA10 protein was detected at the luminal membrane of rat proximal tubule by immunocytochemistry using a polyclonal antibody generated in rabbit. Western blotting also confirmed KCNA10 protein expression in rat cortical membrane. Based on the foregoing we hypothesize KCNA10 is a heteromultimeric, voltage-gated, apical K channel that modulates proximal tubular membrane potential. We therefore, propose to confirm the expression of KCNA10 at the apical membrane of proximal tubules using patch clamp.A knockout mouse model for KCNA10 will be generated and characterized fully, with particular attention paid to its role on proximal tubular function. These studies should provide important information regarding the molecular structure of KCNA10 and its role in renal solute homeostasis.